Epigenetic landscape of the human retina: The characterization of the retinal chromatin landscape will allow the identification of regulatory elements crucial for retinal development and function, including those associated with retinal disease (e.g., AMD genetic variants). We have profiled DNA methylation, open chromatin and histone marks in postmortem adult human retina. In addition, we performed chromatin profiling for the transcription factors NRL and CTCF.The data analysis is in progress. Epigenetic Regulation of Rod Photoreceptor Development and aging: To uncover the role of the epigenome during aging of rod photoreceptors, we generated genome wide profiles of DNA methylation changes in rods at four stages of aging. An integrative analysis that included chromatin landscapes and protein-protein interactions revealed that differentially methylated regions (DMRs) are enriched in dysregulated pathways associated with aging, longevity, synaptic function, and energy homeostasis. Accordingly, we identified mitochondrial respiratory defects in older mouse retinas by using ex vivo mitochondrial oxygen consumption assays. Our studies have uncovered a link between age-related DNA methylation changes and aging pathways including defects in energy homeostasis and revealed genomic features prone to epigenetic changes. Motif enrichment analysis suggest that the binding of the master genome architectural protein CTCF is altered in differentially methylated regions, possibly linking epigenomic changes to gene expression alterations with age. Noncoding Transcriptome of Developing Rods: We have identified several known and novel long noncoding RNAs (lncRNAs), which show dynamic expression during photoreceptor development. Initial data suggests these could be interacting with spliceosomal proteins. To study one of these lncRNAs further, we have generated a gene knockout mouse model. Putative human variants have been identified and are being cloned into expression vectors to study their effect on protein localization and function. To study the role of non-coding RNAs in aging retina, we performed transcriptome analysis in purified rod photoreceptors and identified significantly changed noncoding transcripts. DNA methylation and chromatin profiling of acetylated (H3K27ac) and methylated (H3K27me3) histone 3 revealed differences in chromatin state with age. We are now doing functional analysis of several potentially non-coding RNAs. Evolution of phototransduction genes and the fovea in Vertebrates: Previously, we had shown molecular evidence for the evolution of rod photoreceptors from ancestral cones in mammals. We are now researching this concept further to study the evolution of phototransduction genes in different vertebrate lineages. One of the goals is to study the cellular/molecular changes in the retina concordant with evolutionary shifts between nocturnality and diurnality. This investigation gives us insight into functional changes that allow for night/day transitions. It also reveals how retinas with a variety of rod:cone ratios operate. We are using birds as a study system for understanding the fovea, because birds display a diversity of foveal types. A goal of this project is the investigate the potential of birds as a study system for human macular degeneration. So far, we have collected retinal samples from 14 species of birds and performed transcriptome sequencing. This has generated a huge amount of data that is being analyzed. Determinants of Photoreceptor Pre-Synapse Morphology: Integration of photoreceptors in retinal architecture is essential for visual function. To develop a viable photoreceptor replacement therapy for retinal diseases, we must understand how photoreceptors make specific synaptic connections with inner retinal neurons. We took advantage of the pedicle-like morphology of the terminals in Nrl-/- retina to identify over 600 potential candidate genes that may be associated with determination of presynaptic structures. We have performed a genetic loss of function screen in rods for 72 genes to evaluate their impact on rod pre-synapse morphology. Of these, 26 genes affected the position of terminal within the OPL or the size of the spherule. We experimentally validated the expression of two new proteins, Ywhae and Smarcd3. Cone-cells interactions: Cone photoreceptors which are primary mediators of human vision, interact with different subtypes of bipolar and horizontal cells to transmit visual signals. To investigate the molecular components involved in cone intercellular interactions, we studied the transcriptome of the S-cone like cells in the NRL-/- retina and identified 823 putative genes. To examine their relevance to cone function, we performed a loss of function screen for 19 selected genes in the newborn Nrl-/- mouse retina, with a goal to identify genes associated with cone-cell communication. Based on expression data and putative cellular functions, we have selected 4 genes for further study. Family with Sequence Similarity 19 Member A3 (Fam19a3): Among the genes identified by our screening, we found Fam19a3 which encodes a brain enriched chemokine-like protein. Using a knockin mouse model expressing a FLAG- tagged protein, we detected Fam19a3 expression in cone photoreceptors and type 3b OFF cone bipolar cells. We are now analyzing the function of this protein in retina using knockout mice models. In order to identify Fam19a3 receptor(s), we performed co-immunoprecipitation experiments using the FLAG-tagged mice retina. Ephrin type-A receptor 10 (Epha10): In vivo knockdown of one of the genes, the receptor tyrosine kinase Ephrin type-A receptor 10 (Epha10) altered cone morphology and cell body localization within the retina. We have generated a knockout mouse model to study the function of this gene. Atypical chemokine receptor 1 (Ackr1), Cholecystokinin B receptor (Cckbr): We also obtained mice models for Ackr1 and Cckbr, two other genes identified in our screening, which genes encodes for G protein-coupled receptors. In Vitro 3-D Models of Retinal Development: We are using mouse and human pluripotent stem cell lines to investigate retinal development in vitro. We have used in vivo transcriptome data for molecular staging of organoids and introduced changes in organoid differentiation protocols to facilitate photoreceptor differentiation.